CN109535262A - TrxA-Defensin fusion protein, preparation method and its alexin protein being further prepared and application - Google Patents
TrxA-Defensin fusion protein, preparation method and its alexin protein being further prepared and application Download PDFInfo
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Abstract
The present invention provides a kind of TrxA-Defensin fusion protein, preparation method and its further alexin protein that is prepared and applications, are related to gene engineering technology field.The TrxA-Defensin fusion protein, which is merged by Trx albumen with alexin protein, to be formed.The present invention is by means of protein fusion technology, and by alexin protein gene and Trx gene, i.e., a kind of redox protein thioredoxin Gene Fusion together, expresses that alexin protein in the form of fusion protein.Experiment proves that TrxA-Defensin expressing fusion protein content of the present invention is higher, while by it, further alexin protein antibacterial effect obtained is strong, antibacterial activity is stable, has good prospects for commercial application.TrxA-Defensin fusion protein of the present invention and its further alexin protein obtained can be widely applied among the drug of preparation antibacterium and fungi.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly, to a kind of TrxA-Defensin fusion protein, preparation side
Method and its alexin protein being further prepared and application.
Background technique
Today's society with antibiotic extensive application, pathogenic bacteria drug resistance problems have become medical research heat topic.
How acquisition is screened so that it is the direction that contemporary worker explores that pathogenic bacteria, which will not generate drug resistance antibiotics,.With close
To alexin the study found that alexin (defensin) is become with its unique mechanism of action and molecular characterization is expected to replace over year
For the ideal medicament of conventional antibiotic.
Alexin is applied to production practices, it is also necessary to solve the problems, such as it is many, wherein most important problem is how
Functional active alexin protein is largely obtained, it is general to obtain method there are three types of alexin proteins: (1) directly to be planted from natural move
Alexin protein is extracted in object or microorganism cultures cell；(2) direct chemical synthesis is formed according to the amino acid of alexin protein
And it is modified in vitro according to GAP-associated protein GAP modification with chemical method；(3) alexin protein is expressed with gene engineering method.
Generally in the period of no genetic engineering occurs, the method directly extracted is generallyd use.It can be with although directly extracting
Cheap, safe and reliable alexin protein is obtained, and studies the side often extracted using biology with discovery antibacterial peptide earliest
Method, but since antibacterial peptide is atomic in animals and plants in-vivo content, thus it is natural extract antibacterial peptide often impurity be more, recovery rate compared with
Low, time-consuming length, complex process, therefore the cost is relatively high, cannot achieve large-scale production, this also becomes restriction containing alexin
Antibacterial peptide enters the biggest obstacle of practical application.Although chemically synthesized method is upper possible in production, apply to industry
Change and unrealistic, especially the unstable problem of somewhat expensive, poor efficiency and antibacterial activity to long-chain peptide synthesis, so that it is answered
With being also restricted, above two method is not as good as the development potentiality expressed in vitro by genetic engineering.
Therefore, it researchs and develops a kind of method using genetic engineering and produces and a large amount of and active contain alexin egg
White active antibacterial peptide has great commercial significance, becomes very necessary and urgent.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of TrxA-Defensin fusion protein, the fusion protein by
Trx albumen merges to be formed with alexin protein.Experiment proves that TrxA-Defensin expressing fusion protein content of the present invention compared with
Height, while by it, further alexin protein antibacterial effect obtained is strong, has good prospects for commercial application.
The second object of the present invention is to provide a kind of preparation method of TrxA-Defensin fusion protein, the party
TrxA-Defensin fusion protein is prepared using recombinant DNA technology in method, and this method has extraction preparation efficiency high, obtained
Fusion protein antibacterial activity stablize, while economy is strong, the advantages of being suitable for large-scale industrial production.
The third object of the present invention is to provide alexin protein by above-mentioned TrxA-Defensin fusion protein through enterokinase
Digestion obtains, and alexin protein antibacterial effect is strong, has good prospects for commercial application.
The fourth object of the present invention is to provide a kind of application of alexin protein, and the alexin protein can be widely applied
In antibacterium and fungi activity.
A kind of TrxA-Defensin fusion protein provided by the invention, the fusion protein is by Trx albumen and alexin egg
White fusion is formed；
The amino acid sequence of the TrxA-Defensin fusion protein is as shown in SEQ ID No.1.
Further, the alexin protein is plant defense fibroin, and the amino acid sequence of the alexin protein is such as
Shown in SEQ ID No.3；
Preferably, the plant defense fibroin is expressed to obtain by comospore poplar phylaxin gene, the comospore poplar alexin
Gene is as shown in SEQ ID No.4.
Further, the TrxA-Defensin fusion protein is solvable state.
A kind of preparation method of TrxA-Defensin fusion protein provided by the invention, the preparation method includes following
Step:
Prepare the recombinant DNA comprising alexin protein gene and Trx gene first, then by recombinant DNA molecules convert by
Body cell, culture obtain TrxA-Defensin fusion protein.
Further, the recipient cell is the bacterium for generating fusion protein.
Further, the preparation method specifically includes the following steps:
(a): first extraction comospore poplar total serum IgE and reversion rate be the first chain cDNA, and using the first chain cDNA as template into
Row PCR amplification, as shown in SEQ ID No.5, downstream primer sequence obtains upstream primer sequence as shown in SEQ ID No.6
Comospore poplar phylaxin gene；
(b): the method for the comospore poplar phylaxin gene PCR that step (a) is obtained introduce restriction enzyme BamHI and
The restriction enzyme site of NotI, upstream primer sequence is as shown in SEQ ID No.7, and downstream primer sequence is as shown in SEQ ID No.8；
The PCR product of recycling and PET-32a plasmid vector are then subjected to digestion with two kinds of restriction enzymes of BamHI and NotI；So
Afterwards under the action of T4DNA Ligase ligase, plasmid vector and gene are connected, recombinant DNA molecules are obtained；
(c): by recombinant DNA molecules transformed competence colibacillus bacterium, culture obtains TrxA-Defensin fusion protein.
Further, the bacterium in the step (c) is bacillus coli DH 5 alpha or e. coli bl21 (DE3).
Further, the preparation method further includes the steps that isolating and purifying fusion protein；
Preferably, Ni is used in the purification procedures+- NTA Purification Resin post separation removes foreign protein.
A kind of alexin protein provided by the invention, the alexin protein is by above-mentioned TrxA-Defensin fusion protein
It is obtained through enterokinase digestion.
A kind of application of the above-mentioned alexin protein provided by the invention in preparation antibacterium and fungi activity drug.
Compared with prior art, the invention has the benefit that
TrxA-Defensin fusion protein provided by the invention, the fusion protein are melted by Trx albumen with alexin protein
It closes and is formed.The present invention is by means of protein fusion technology, by alexin protein gene and Trx gene, i.e., a kind of redox protein
Thioredoxin Gene Fusion together, expresses that alexin protein in the form of fusion protein.Experiment proves that this
It is higher to invent TrxA-Defensin expressing fusion protein content, while by its further alexin protein antibacterial effect obtained
By force, antibacterial activity is stablized, and has good prospects for commercial application.
The preparation method of TrxA-Defensin fusion protein provided by the invention, prepares first comprising alexin protein base
The recombinant DNA of cause and Trx gene, then by recombinant DNA molecules transformed acceptor cell, culture obtains TrxA-Defensin fusion
Albumen.TrxA-Defensin fusion protein is prepared using recombinant DNA technology in above-mentioned preparation method, and this method, which has, to be extracted
The advantages of preparation efficiency is high, fusion protein obtained is activity stabilized, while economy is strong, is suitable for large-scale industrial production.
Above-mentioned alexin protein provided by the invention, the alexin protein is by above-mentioned TrxA-Defensin fusion protein
It obtains, is can be widely applied among the drug of preparation antibacterium and fungi activity through enterokinase digestion.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the Information Atlas for the PET-32a plasmid vector that embodiment 2 provides in the present invention；
Fig. 2 is 12%SDS-PAGE after the fusion protein TrxA-Defensin inducing expression that the embodiment of the present invention 3 provides
Figure；
Fig. 3 is that the fusion protein TrxA-Defensin that the embodiment of the present invention 3 provides purifies 12%SDS-PAGE gel electrophoresis
Figure and western blot verify TrxA-Defensin fusion protein figure；
Fig. 4 is the enterokinase digestion TrxA label that the embodiment of the present invention 4 provides and the 4-20% gradient for obtaining Defensin
SDS-PAGE electrophoresis；
Fig. 5 is the anti-large intestine bar of alexin Defensin albumen after enterokinase digestion that test example 1 of the present invention provides
Bacterium K12D31 and the antibacterial loop graph of Agrobacterium EHA105；
Fig. 6 is the antimycotic antibacterial loop graph of the Defensin albumen after enterokinase digestion that test example 2 of the present invention provides.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
According to an aspect of the present invention, a kind of TrxA-Defensin fusion protein, the fusion protein is by Trx albumen
It merges to be formed with alexin protein；
The amino acid sequence of the TrxA-Defensin fusion protein is as shown in SEQ ID No.1.
TrxA-Defensin fusion protein provided by the invention, the fusion protein are melted by Trx albumen with alexin protein
It closes and is formed.The present invention is by means of protein fusion technology, by alexin protein gene and Trx gene, i.e., a kind of redox protein
Thioredoxin Gene Fusion together, expresses that alexin protein in the form of fusion protein.Experiment proves that this
It is higher to invent TrxA-Defensin expressing fusion protein content, while by its further alexin protein antibacterial effect obtained
By force, activity stabilized, there is good prospects for commercial application.The amino acid sequence such as SEQ of the TrxA-Defensin fusion protein
Shown in ID No.1.
Preferably, the nucleotide sequence of the TrxA-Defensin fusion protein is as shown in SEQ ID No.2.
In the preferred embodiment of the present invention, the alexin protein is plant defense fibroin, the defence
The amino acid sequence of fibroin is as shown in SEQ ID No.3；
As a preferred embodiment, above-mentioned alexin protein is plant defense fibroin.Nineteen ninety by
Mendez etc. measures its three-dimensional structure with NMR spectroscopy from wheat, barley seed plant defense fibroin isolated first,
It was found that it is similar to mammal and insect defensin, all have sterilization well and antiviral effect.
Preferably, the plant defense fibroin is expressed to obtain by comospore poplar phylaxin gene, the comospore poplar alexin
Gene is as shown in SEQ ID No.4.
In the preferred embodiment of the present invention, the TrxA-Defensin fusion protein is solvable state.
As a preferred embodiment, above-mentioned TrxA-Defensin fusion protein is solvable state.The present invention by
In protein fusion technology, comospore poplar phylaxin gene and TRX gene, i.e., a kind of redox protein thioredoxin gene
It is fused together, expresses that comospore poplar alexin in the form of fusion protein, as a result the fusion protein is common various
Solubility expression is respectively formed under condition of culture, expression contents are higher while being had by its further alexin protein obtained non-
Often good antibacterial activity.
According to an aspect of the present invention, a kind of preparation method of TrxA-Defensin fusion protein, the preparation method
The following steps are included:
Prepare the recombinant DNA comprising alexin protein gene and Trx gene first, then by recombinant DNA molecules convert by
Body cell, culture obtain TrxA-Defensin fusion protein.
Currently used is recombinant DNA technology, generally comprises four steps: 1. obtaining target gene；2. it is connect with cloning vector,
Form new recombinant DNA molecules；3. with recombinant DNA molecules transformed acceptor cell, and can be replicated in recipient cell and heredity；④
To transformant screening and identification.5. obtaining required inhereditary feature by culture to the cell or organism for obtaining foreign gene
Or give expression to required product.
The preparation method of TrxA-Defensin fusion protein provided by the invention, prepares first comprising alexin protein base
The recombinant DNA of cause and Trx gene, then by recombinant DNA molecules transformed acceptor cell, culture obtains TrxA-Defensin fusion
Albumen.TrxA-Defensin fusion protein is prepared using recombinant DNA technology in above-mentioned preparation method, and this method, which has, to be extracted
The advantages of preparation efficiency is high, fusion protein obtained is activity stabilized, while economy is strong, is suitable for large-scale industrial production.
In the preferred embodiment of the present invention, the recipient cell is the bacterium for generating fusion protein.
As a preferred embodiment, above-mentioned recipient cell is the bacterium for generating fusion protein.Preferably, described thin
Bacterium is bacillus coli DH 5 alpha or e. coli bl21 (DE3).
In the preferred embodiment of the present invention, the preparation method specifically includes the following steps:
(a): first extraction comospore poplar total serum IgE and reversion rate be the first chain cDNA, and using the first chain cDNA as template into
Row PCR amplification, as shown in SEQ ID No.5, downstream primer sequence obtains upstream primer sequence as shown in SEQ ID No.6
Comospore poplar phylaxin gene；
(b): the method for the comospore poplar phylaxin gene PCR that step (a) is obtained introduce restriction enzyme BamHI and
The restriction enzyme site of NotI, upstream primer sequence is as shown in SEQ ID No.7, and downstream primer sequence is as shown in SEQ ID No.8；
The PCR product of recycling and PET-32a plasmid vector are then subjected to digestion with two kinds of restriction enzymes of BamHI and NotI；So
Afterwards under the action of T4DNA Ligase ligase, plasmid vector and gene are connected, recombinant DNA molecules are obtained；
(c): by recombinant DNA molecules transformed competence colibacillus bacterium, culture obtains TrxA-Defensin fusion protein.
In above-mentioned preferred embodiment, the bacterium in the step (c) is bacillus coli DH 5 alpha or e. coli bl21
(DE3)。
In the preferred embodiment of the present invention, the preparation method further includes isolating and purifying fusion protein
The step of；
As a preferred embodiment, above-mentioned preparation method further includes the step for isolating and purifying fusion protein
Suddenly, specifically: will culture obtain the ultrasonication on ice of TrxA-Defensin fusion protein, 4 DEG C, 12000rpm, 20min from
After heart ultrasonication liquid, precipitating is abandoned, stays supernatant to cross Purification Resin column and washes away foreign protein, after collecting the degerming of desired polypeptides diafiltration
It saves.
Preferably, Ni is used in the purification procedures+- NTA Purification Resin post separation removes foreign protein.
As a preferred embodiment, above-mentioned purification procedures are removed using Ni+-NTA Purification Resin post separation
Foreign protein.
Preferably, the Ni+-NTA Purification Resin post separation removes the elution step of foreign protein are as follows: uses Binding
After Buffer balances Ni+-NTA column, supernatant of the loading containing soluble recombinant polypeptide after end of the sample, first uses Binding
Buffer is tentatively eluted, and then, then with Washing buffer washes away albumen, finally elutes mesh with Eluent buffer
Albumen.
It is furthermore preferred that the composition of the Binding Buffer are as follows: 20mmol/L Imidazole, 0.5mol/L NaCl,
20mmol/L Tris HCl, pH 8.0.
The composition of Washing buffer are as follows: 50mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L Tris
HCl, pH 8.0.
The composition of Eluent buffer are as follows: 250mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L Tris
HCl, pH 8.0.
According to an aspect of the present invention, a kind of alexin protein, the alexin protein is by above-mentioned TrxA-Defensin
Fusion protein is obtained through enterokinase digestion.
According to an aspect of the present invention, a kind of above-mentioned alexin protein is in preparation antibacterium and fungi activity drug
Using.
Above-mentioned alexin protein provided by the invention, the alexin protein can be widely applied to preparation antibacterium and fungi
Among active drug.
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail.
Embodiment 1
Clone poplar phylaxin gene:
(1) total serum IgE of comospore poplar is extracted according to its operation manual using RNA extracts kit (Takara), and passes through 2%
Agarose gel electrophoresis identifies its purity, and ultraviolet specrophotometer measures its concentration.Using Takara cDNA Reverse Transcriptase kit
It is transcribed into the first chain cDNA.
(2) design of primers: the poplar phylaxin gene aligning primer announced according to NCBI:
Forward primer1 sequence: 5 '-ATGGAGATCAAGAGATCCTTTGG-3 ' (sequence such as SEQ ID No.5 institute
Show),
Reverse primer1 sequence: 5 '-TTAACAAAGCTTGGTGCAAA AAC-3 ' (sequence such as SEQ ID No.6
It is shown)；
(3) PCR: being that template carries out PCR using the resulting first chain cDNA of reverse transcription, after the reaction was completed by product in 1%
It is separated by electrophoresis in Ago-Gel, is recycled to obtain DNA band with plastic recovery kit, PETASY-T3 carrier is cloned into, through containing
The agarose plate of Amp antibiotic (50mg/mL) screens transformant, according to color reaction choose positive colony be sent to Nanjing gold this
Auspicious sequencing company carries out base sequence measurement, obtains the segment of 228bp, and the sequence is consistent with the sequence alignment that NCBI is announced,
It is thus determined that the sequence is comospore poplar phylaxin gene sequence, (sequence is as shown in SEQ ID No.4)
In the comospore poplar phylaxin gene sequence (sequence is as shown in SEQ ID No.4), 1-27 base is the gene
5 ' UTR noncoding regions, 28-256 base is the area complete encoding sequence ORF of the gene, translates 75 amino acid, 257-
No. 614 bases are 3 ' UTR noncoding regions of the gene.
The reaction system of above-mentioned PCR reaction are as follows: each 2 μ L, 10mmol/L dNTP of the 10 μm of upstream and downstream ol/L primers 1 μ L, 10
5 μ L, cDNA template of × LaTaq buffer, 3 μ L, LaTaq enzyme, 0.5 μ L, adds ddH2O to complement to 50 μ L.
The response procedures of above-mentioned PCR reaction are as follows: 95 DEG C of 10min, (95 DEG C of 40s, 58 DEG C of 40s, 72 DEG C of 40s), 35 circulations,
Last 72 DEG C of extensions 10min.
Embodiment 2
The building of recombinant DNA molecules:
(1) method for the comospore poplar phylaxin gene PCR for obtaining above-described embodiment 1 introduces restriction enzyme
The restriction enzyme site of BamHI and NotI, upstream primer sequence is as shown in SEQ ID No.7, downstream primer sequence such as SEQ ID
Shown in No.8；
(2) by PCR product after the recovery and PET-32a plasmid vector with two kinds of restriction enzymes of BamHI and NotI into
Row digestion；Then under the action of T4DNA Ligase ligase, plasmid vector and gene is connected, recombinant DNA molecules are obtained.
As shown in FIG. 1, FIG. 1 is PET-32a plasmid vector maps.
It is to contain TRX gene order using its upstream that the present embodiment, which selects pET-32a plasmid vector,；It can certainly be
TRX gene is inserted in PCR method on the carrier that other contain comospore poplar phylaxin gene sequence, can equally realize comospore poplar
The purpose of phylaxin gene expressing fusion protein clone's building.
Embodiment 3
The vivoexpression of TrxA-Defensin fusion protein and purifying:
The recombinant DNA molecules transformed competence colibacillus e. coli bl21 (DE3) that embodiment 2 is obtained obtains and contains PET-32a-
Defensin recombinant bacterial strain, after from picking single colonie on LB plate, the single colonie of picking contains 50 mcg/mls at 4 milliliters
It is incubated overnight in LB liquid medium, overnight culture is then trained in the LB liquid that the ratio of 1:250 is added to 2 500 milliliters
It supports in base, in IPTG concentration 1.0mmol/L, 16 DEG C, 18 hours receipts bacterium of inducing expression, on ice ultrasonication under conditions of 110rpm
Thallus is expressed, 4 DEG C, after 12000rpm, 20min are centrifuged ultrasonication liquid, precipitating are abandoned, stays supernatant to cross Ni+-NTA column and wash away miscellaneous egg
It is white, it is saved after collecting the diafiltration degerming of TrxA-Defensin fusion protein；
Above-mentioned Ni+-NTA Purification Resin post separation removes the elution step of foreign protein are as follows: flat using Binding Buffer
It weighs after Ni+-NTA column, supernatant of the loading containing soluble recombinant polypeptide, after end of the sample, first at the beginning of Binding Buffer is carried out
Step elution, then, then with Washing buffer washes away albumen, finally elutes destination protein with Eluent buffer.
Wherein, the composition of the Binding Buffer are as follows: 20mmol/L Imidazole, 0.5mol/L NaCl,
20mmol/L Tris HCl, pH 8.0.
The composition of Washing buffer are as follows: 50mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L Tris
HCl, pH 8.0.
The composition of Eluent buffer are as follows: 250mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L Tris
HCl, pH 8.0.
As shown in Fig. 2, Fig. 2 is 12%SDS-PAGE figure after fusion protein TrxA-Defensin inducing expression, wherein M is
Marker, swimming lane 1 are not induce, and swimming lane 2-9 is different monoclonal inducing expressions.
As shown in figure 3, Fig. 3 be fusion protein TrxA-Defensin purify 12%SDS-PAGE gel electrophoresis figure and
Western blot verifies TrxA-Defensin fusion protein figure, in which:
M is Marker, and swimming lane 1 is inducing expression, and swimming lane 2 is inducing expression precipitating, and swimming lane 3 is inducing expression supernatant, swimming
Road 4 is sample solution, and swimming lane 5 is sample solution, and swimming lane 6 is efflux, and swimming lane 7,8 is eluent, and swimming lane 9 is that western blot is tested
Card.
Embodiment 4
Embodiment 3 is purified into obtained TrxA-Defensin fusion protein in the presence of enterokinase (10U), at 25 DEG C
It is incubated for 16 hours, solution is added to again in the nickel column of the resin containing Ni-IDA, remove the TrxA of its label.Do not had finally
The alexin protein of Trx label.
As shown in figure 4, Fig. 4 is enterokinase digestion TrxA label and the 4-20% gradient SDS-PAGE electricity for obtaining Defensin
Swimming figure, in which:
M is Marker, and swimming lane 1 is the TrxA-Defensin of purifying, and swimming lane 2 is by enterokinase digestion, and swimming lane 3 is warp
The alexin protein of NI column elution efflux.
Test example 1
Activity identification of the alexin protein made from embodiment 4 in anti-Escherichia coli and Agrobacterium:
(1) alexin (Defensin) albumen is in anti-e. coli k12 D31 activity identification
Using hole diffusion method measurement alexin alexin Defensin albumen to the antibacterial activity of e. coli k12 D31.
E. coli k12 D31 is inoculated in LB liquid medium, 37 DEG C of 220rpm are cultivated 6 hours, added to and melt by 1% bacterium amount
In the solid LB media of change (35-50 DEG C), sterile disposable plate is laid on by 30mL/ ware after mixing.After solidification, in sterile item
It is punched under part, initial concentration made from the embodiment of the present invention 4 is 96 μ g/ml alexin Defensin eggs according to doubling dilution by addition
It is white to be diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 37 DEG C of incubators 24 hours, observes bacteriostatic activity, knot
Fruit sees Fig. 5 A, has apparent anti-e. coli k12 D31 activity.
Its result is as shown in Figure 5A, and Fig. 5 A is the anti-e. coli k12 D31 of Defensin albumen after enterokinase digestion
Antibacterial loop graph, in which:
C is 120 μ l 0.9%NaCl (negative control), and 1 is 120 μ l, 96 μ g/ml alexin protein, and 2 be 120 μ l, 48 μ
G/ml alexin protein, 3 be 120 μ l, 24 μ g/ml alexin protein, and 4 be 120 μ l12 μ g/ml alexin proteins, and 5 be 120 μ l
6 μ g/ml alexin proteins, 6 be 120 μ l, 3 μ g/ml alexin protein, and 7 be 120 μ l, 1.5 μ g/ml alexin protein.
(2) alexin (Defensin) albumen is in anti-Agrobacterium EHA105 activity identification
Using hole diffusion method measurement alexin Defensin albumen to the antibacterial activity of Agrobacterium EHA105.
Agrobacterium EHA105 is inoculated in LB liquid medium, 28 DEG C of 220rpm are cultivated 12 hours, add to thawing by 1% bacterium amount
Solid LB media in (35-50 DEG C), be laid on sterile disposable plate by 30mL/ ware after mixing.After solidification, in aseptic condition
Initial concentration made from the embodiment of the present invention 4 is 96 μ g/ml alexin Defensin albumen according to doubling dilution by lower punching, addition
7 parts are diluted to, and every hole is added 120 μ l alexin proteins and is placed on 28 DEG C of incubators 48 hours, observes bacteriostatic activity, as a result
See Fig. 5 B, there is apparent anti-Agrobacterium EHA105 activity.
Its result is as shown in Figure 5 B, and Fig. 5 B is the anti-Agrobacterium EHA105 suppression of Defensin albumen after enterokinase digestion
Bacterium loop graph, in which:
C is 120 μ l0.9%NaCl (negative control), and 1 is 120 μ l, 96 μ g/ml alexin protein, and 2 be 120 μ l, 48 μ g/
Ml alexin protein, 3 be 120 μ l24 μ g/ml alexin proteins, and 4 be 120 μ l12 μ g/ml alexin proteins, and 5 be 120 μ l, 6 μ
G/ml alexin protein, 6 be 120 μ l, 3 μ g/ml alexin protein, and 7 be 120 μ l, 1.5 μ g/ml alexin protein.
Test example 2
The antimycotic activity identification of alexin made from embodiment 4 (Defensin) albumen:
(1) anti-aspergillus niger Aspergillus niger activity identification
It is living using antibacterial of the diffusion method measurement alexin Defensin albumen in hole to aspergillus niger Aspergillus niger
Property.
Aspergillus nigers is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, by 1% bacterium amount
(35-50 DEG C) is added in the potato culture medium of thawing, is laid on sterile disposable plate by 30mL/ ware after mixing.After solidification, in nothing
It is punched under the conditions of bacterium, initial concentration made from the embodiment of the present invention 4 is 40 μ g/ml alexins according to doubling dilution by addition
Defensin albumen is diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes
As a result bacteriostatic activity is shown in Fig. 6 A, have apparent antifungal activity.
(2) anti-alternaric bacteria Alternaria Nee activity identification
It is living using antibacterial of the diffusion method measurement alexin Defensin albumen in hole to alternaric bacteria Alternaria Nee
Property.
Alternaria Nee is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, added to by 1% bacterium amount
In the potato culture medium of thawing (35-50 DEG C), sterile disposable plate is laid on by 30mL/ ware after mixing.After solidification, in sterile item
It is punched under part, initial concentration made from the embodiment of the present invention 4 is 40 μ g/ml alexin Defensin eggs according to doubling dilution by addition
It is white to be diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes bacteriostatic activity, knot
Fruit sees Fig. 6 B, has apparent antifungal activity.
(3) activity identification of anti-mucor Mucor corymbifer.
It is living using antibacterial of the diffusion method measurement alexin Defensin albumen in hole to mucor Mucor corymbifer.
Property.
Mucor corymbifer is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, added by 1% bacterium amount
Into the potato culture medium of thawing (35-50 DEG C), sterile disposable plate is laid on by 10mL/ ware after mixing.After solidification, sterile
Under the conditions of punch, be added according to doubling dilution by initial concentration made from the embodiment of the present invention 4 be 40 μ g/ml alexin Defensin
Albumen is diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes bacteriostatic activity,
As a result see Fig. 6 C, there is apparent antifungal activity.
(4) activity identification of anti-poplar disk single lattice spore bacterium Marssonina populi.
Using hole diffusion method measurement alexin TrxA-Defensin albumen to poplar disk single lattice spore bacterium Marssonina
Populi. antibacterial activity.
Marssonina populi. is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, by 1% bacterium amount
(35-50 DEG C) is added in the potato culture medium of thawing, is laid on sterile disposable plate by 30mL/ ware after mixing.After solidification, in nothing
It is punched under the conditions of bacterium, initial concentration made from the embodiment of the present invention 4 is 40 μ g/ml alexins according to doubling dilution by addition
Defensin albumen is diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes
As a result bacteriostatic activity is shown in Fig. 6 D, have apparent antifungal activity.
(5) activity identification of anti-rhizopus Rhizopus sp.
It is living to rhizopus Rhizopus sp. antibacterial using hole diffusion method measurement alexin TrxA-Defensin albumen
Property.
Rhizopus sp. is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, added to and melt by 1% bacterium amount
In the potato culture medium of change (35-50 DEG C), sterile disposable plate is laid on by 30mL/ ware after mixing.After solidification, in aseptic condition
Initial concentration made from the embodiment of the present invention 4 is 40 μ g/ml alexin Defensin albumen according to doubling dilution by lower punching, addition
7 parts are diluted to, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes bacteriostatic activity, as a result
See Fig. 6 E, there is apparent antifungal activity.
(6) activity identification of anti-neurospora crassa Neurospora crassa
Neurospora crassa Neurospora crassa is resisted using hole diffusion method measurement alexin Defensin albumen
Bacterium activity.
Neurospora crassa is inoculated in liquid potato culture medium, 25 DEG C of 180rpm are cultivated 6 hours, added by 1% bacterium amount
Into the potato culture medium of thawing (35-50 DEG C), sterile disposable plate is laid on by 30mL/ ware after mixing.After solidification, sterile
Under the conditions of punch, be added according to doubling dilution by initial concentration made from the embodiment of the present invention 4 be 40 μ g/ml alexin Defensin
Albumen is diluted to 7 parts, and every hole is added 120 μ l alexin proteins and is placed on 25 DEG C of incubators 72 hours, observes bacteriostatic activity,
As a result see Fig. 6 F, there is apparent antifungal activity.
Fig. 6 is the antimycotic antibacterial loop graph of the Defensin albumen after enterokinase digestion that test example 2 provides.Wherein:
C is 120 μ l 0.9%NaCl (negative control), and 1 is 120 μ l, 100 μ g/ml alexin protein, and 2 be 120 μ l, 50 μ
G/ml alexin protein, 3 be 120 μ l25 μ g/ml alexin proteins, and 4 be 120 μ l, 12.5 μ g/ml alexin protein, and 5 be 120 μ
6.5 μ g/ml alexin protein of l, 6 be 120 μ l3.75 μ g/ml alexin proteins, and 7 be 120 μ l, 1.85 μ g/ml alexin egg
It is white.
In conclusion TrxA-Defensin fusion protein provided by the invention, the fusion protein is by Trx albumen and prevents
Imperial fibroin merges to be formed.The present invention is by means of protein fusion technology, by alexin protein gene and Trx gene, i.e., a kind of oxygen
Change reduction albumen thioredoxin Gene Fusion together, expresses that alexin protein in the form of fusion protein.Through trying
Verifying, TrxA-Defensin expressing fusion protein content of the present invention is higher, and fusion protein obtains anti-after enterokinase digestion
Imperial element (Defensin albumen) antibacterial effect is strong, has good prospects for commercial application.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Nanjing Forestry University
<120>it TrxA-Defensin fusion protein, preparation method and its alexin protein being further prepared and answers
With
<130> 4687461313
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 242
<212> PRT
<213>TrxA-Defensin fusion protein
<400> 1
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
115 120 125
Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
130 135 140
His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
145 150 155 160
Ala Asp Ile Gly Ser Glu Phe Met Glu Ile Lys Arg Ser Phe Gly Leu
165 170 175
Phe Phe Leu Leu Leu Ile Val Leu Ala Ser Gln Glu Val Val Val Pro
180 185 190
Thr Glu Ala Arg Val Cys Gln Ser Gln Ser His Tyr Phe Lys Gly Pro
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Cys Ala Arg Asp His Asn Cys Ala Trp Val Cys Arg Asn Glu Gly Phe
210 215 220
Ser Gly Gly Arg Cys Lys Gly Phe Arg Arg Arg Cys Phe Cys Thr Lys
225 230 235 240
Leu Cys
<210> 2
<211> 729
<212> DNA
<213>TrxA-Defensin fusion protein
<400> 2
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120
ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300
aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360
catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420
ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480
gctgatatcg gatccgaatt catggagatc aagagatcct ttgggctttt cttcttgctc 540
ctcattgtct tggcttctca agaggtggtg gtgcctactg aggcaagggt ttgccagtca 600
cagagccatt attttaaagg cccatgtgct agggaccata actgtgcatg ggtgtgcagg 660
aatgaaggtt tctctggggg aagatgcaaa gggttccgtc gccgctgttt ttgcaccaag 720
ctttgttaa 729
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<213>comospore poplar alexin protein
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1 5 10 15
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20 25 30
Ser Gln Ser His Tyr Phe Lys Gly Pro Cys Ala Arg Asp His Asn Cys
35 40 45
Ala Trp Val Cys Arg Asn Glu Gly Phe Ser Gly Gly Arg Cys Lys Gly
50 55 60
Phe Arg Arg Arg Cys Phe Cys Thr Lys Leu Cys
65 70 75
<210> 4
<211> 600
<212> DNA
<213>comospore poplar phylaxin gene
<400> 4
agcaaacgtt ttaagcaaac taaagacatg gagatcaaga gatcctttgg gcttttcttc 60
ttgctcctca ttgtcttggc ttctcaagag gtggtggtgc ctactgaggc aagggtttgc 120
cagtcacaga gccattattt taaaggccca tgtgctaggg accataactg tgcatgggtg 180
tgcaggaatg aaggtttctc tgggggaaga tgcaaagggt tccgtcgccg ctgtttttgc 240
accaagcttt gttaagttaa gcaaagaaaa tgcatcatgt gttaaggttg ccaagcatgc 300
attttgacat gaaaaaagca ctatcttaat atcctcttgt tttttgttca attagctgag 360
tatccataag aatgtcagca tcacttgtgt tagcttgctc ttcagctaga caacttgcta 420
gctttagaac ctgctgctga tcgatgatgc tggggttaat gtcaatgttg gtttcgttat 480
gtaataataa actctccctt cttgttcgag gaagcattct tgtcacctaa tttctggtgt 540
tctcaatgga ttcttgatga gaaaatgtga gtgatggcaa agagtccaaa cacaacaaga 600
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<213>artificial sequence
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atggagatca agagatcctt tgg 23
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<211> 23
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<213>artificial sequence
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ttaacaaagc ttggtgcaaa aac 23
<210> 7
<211> 29
<212> DNA
<213>artificial sequence
<400> 7
cgggatccat ggagatcaag agatccttt 29
<210> 8
<211> 39
<212> DNA
<213>artificial sequence
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aaggaaaaaa gcggccgctt aacaaagctt ggtgcaaaa 39
Claims (10)
1. a kind of TrxA-Defensin fusion protein, which is characterized in that the fusion protein is by Trx albumen and alexin protein
Fusion is formed；
The amino acid sequence of the TrxA-Defensin fusion protein is as shown in SEQ ID No.1.
2. TrxA-Defensin fusion protein according to claim 1, which is characterized in that the alexin protein is to plant
Object alexin protein, the amino acid sequence of the alexin protein is as shown in SEQ ID No.3；
Preferably, the plant defense fibroin is expressed to obtain by comospore poplar phylaxin gene, the comospore poplar phylaxin gene
As shown in SEQ ID No.4.
3. TrxA-Defensin fusion protein according to claim 1, which is characterized in that the TrxA-Defensin melts
Hop protein is solvable state.
4. a kind of preparation method of TrxA-Defensin fusion proteins described in any item according to claim 1~3, feature
It is, the preparation method comprises the following steps:
The recombinant DNA comprising alexin protein gene and Trx gene is prepared first, it is then that recombinant DNA molecules transformation receptor is thin
Born of the same parents, culture obtain TrxA-Defensin fusion protein.
5. the preparation method of TrxA-Defensin fusion protein according to claim 4, which is characterized in that the receptor
Cell is the bacterium for generating fusion protein.
6. the preparation method of TrxA-Defensin fusion protein according to claim 4, which is characterized in that the preparation
Method specifically includes the following steps:
(a): the total serum IgE and reversion rate of extraction comospore poplar first are the first chain cDNA, and carry out PCR by template of the first chain cDNA
Amplification, for upstream primer sequence as shown in SEQ ID No.5, downstream primer sequence obtains comospore poplar as shown in SEQ ID No.6
Phylaxin gene；
(b): the method for the comospore poplar phylaxin gene PCR that step (a) is obtained introduces restriction enzyme BamHI and NotI
Restriction enzyme site, upstream primer sequence is as shown in SEQ ID No.7, and downstream primer sequence is as shown in SEQ ID No.8；Then
The PCR product of recycling and PET-32a plasmid vector are subjected to digestion with two kinds of restriction enzymes of BamHI and NotI；Then exist
Under the action of T4DNA Ligase ligase, plasmid vector and gene are connected, recombinant DNA molecules are obtained；
(c): by recombinant DNA molecules transformed competence colibacillus bacterium, culture obtains TrxA-Defensin fusion protein.
7. the preparation method of TrxA-Defensin fusion protein according to claim 6, which is characterized in that the step
(c) bacterium in is bacillus coli DH 5 alpha or e. coli bl21 (DE3).
8. the preparation method of TrxA-Defensin fusion protein according to claim 4, which is characterized in that the preparation
Method further includes the steps that isolating and purifying fusion protein；
Preferably, Ni is used in the purification procedures+- NTA Purification Resin post separation removes foreign protein.
9. a kind of alexin protein, which is characterized in that the alexin protein is by the described in any item TrxA- of claims 1 to 3
Defensin fusion protein is obtained through enterokinase digestion.
10. a kind of application of alexin protein according to claim 9 in preparation antibacterium and fungi activity drug.
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| CN114249839A (en) * | 2021-12-31 | 2022-03-29 | 山东林森生物制品股份有限公司 | Fusion protein of type III collagen, expression system, pharmaceutical composition and application |
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